LKB1 tumor suppressor regulates AMP kinase/mTOR-independent cell growth and proliferation via the phosphorylation of Yap.

The liver kinase B1 (LKB1) tumor suppressor inhibits cell growth through its regulation of cellular metabolism and apical-basal polarity. The best understood mechanism whereby LKB1 limits cell growth is through activation of the AMP-activated-protein-kinase/mammalian-target-of-rapamycin (AMPK/mTOR) pathway to control metabolism. As LKB1 is also required for polarized epithelial cells to resist hyperplasia, it is anticipated to function through additional mechanisms. Recently, Yes-associated protein (Yap) has emerged as a transcriptional co-activator that modulates tissue homeostasis in response to cell-cell contact. Thus this study examined a possible connection between Yap and LKB1. Restoration of LKB1 expression in HeLa cells, which lack this tumor suppressor, or short-hairpin RNA knockdown of LKB1 in NTERT immortalized keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). However, failure of Lats1/2 knockdown to suppress LKB1-mediated Yap regulation suggested that LKB1 signals to Yap via a non-canonical pathway. Additionally, LKB1 inhibited Yap independently of either AMPK or mTOR activation. These findings reveal a novel mechanism whereby LKB1 may restrict cancer cell growth via the inhibition of Yap.

The insert region of RhoA is essential for Rho kinase activation and cellular transformation.

RhoA is involved in multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These processes are mediated by a variety of downstream effector proteins. However, which effectors are involved in cellular transformation and how these proteins are activated following interaction with Rho remains to be established. A unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region, which may confer Rho-specific signaling events. Here we report that deletion of the insert region does not result in impaired effector binding. Instead, this insert deletion mutant (RhoDeltaRas, in which the insert helix has been replaced with loop 8 of Ras) acted in a dominant inhibitory fashion to block RhoA-induced transformation. Since RhoDeltaRas failed to promote stress fiber formation, we examined the ability of this mutant to bind to and subsequently activate Rho kinase. Surprisingly, RhoDeltaRas-GTP coprecipitated with Rho kinase but failed to activate it in vivo. These data suggested that the insert domain is not required for Rho kinase binding but plays a role in its activation. The constitutively active catalytic domain of Rho kinase did not promote focus formation alone or in the presence of Raf(340D) but cooperated with RhoDeltaRas to induce cellular transformation. This suggests that Rho kinase needs to cooperate with additional Rho effectors to promote transformation. Further, the Rho kinase catalytic domain reversed the inhibitory effect of RhoDeltaRas on Rho-induced transformation, suggesting that one of the downstream targets of Rho-induced transformation abrogated by RhoDeltaRas is indeed Rho kinase. In conclusion, we have demonstrated that the insert region of RhoA is required for Rho kinase activation but not for binding and that this kinase activity is required to induce morphologic transformation of NIH 3T3 cells.

Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A.

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.

Analysis of function and regulation of proteins that mediate signal transduction by use of lipid-modified plasma membrane-targeting sequences.

It is now established that the function of many signaling molecules is controlled, in part, by regulation of subcellular localization. For example, the dynamic recruitment of normally cytosolic proteins to the plasma membrane, by activated Ras or activated receptor tyrosine kinases, facilitates their interaction with other membrane-associated components that participate in their full activation (e.g., Raf-1). Therefore, the creation of chimeric proteins that contain lipid-modified signaling sequences that direct membrane localization allows the generation of constitutively activated variants of such proteins. The amino-terminal myristoylation signal sequence of Src family proteins and the carboxy-terminal prenylation signal sequence of Ras proteins have been widely used to achieve this goal. Such membrane-targeted variants have proved to be valuable reagents in the study of the biochemical and biological properties of many signaling molecules.

Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction.

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral.

Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.

Pancreatic eukaryotic initiation factor-2alpha kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress.

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). Recently, we identified a new family member, pancreatic eIF-2alpha kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2alpha phosphorylation was coincident with increased PEK autophosphorylation and eIF-2alpha kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2alpha and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2alpha kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.

M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6.

M-Ras is a Ras-related protein that shares approximately 55% identity with K-Ras and TC21. The M-Ras message was widely expressed but was most predominant in ovary and brain. Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors. Although the mitogen-activated protein kinase/ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation. These data indicate that multiple pathways must contribute to M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase delta, RalGDS, and Rin1. Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.

Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation.

Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the "insert domain"(residues 123-137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75-92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivo studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.

Identification of Grb4/Nckbeta, a src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck.

Adapter proteins made up of Src homology (SH) domains mediate multiple cellular signaling events initiated by receptor protein tyrosine kinases. Here we report that Grb4 is an adapter protein closely related to but distinct from Nck that is made up of three SH3 domains and one SH2 domain. Northern analysis indicated that both genes are expressed in multiple tissues. Both Nck and Grb4 proteins could associate with receptor tyrosine kinases and the SH3-binding proteins PAK, Sos1, and PRK2, and they synergized with v-Abl and Sos to induce gene expression via the transcription factor Elk-1. Although neither protein was transforming on its own, both Nck and Grb4 cooperated with v-Abl to transform NIH 3T3 cells and influenced the morphology and anchorage-dependent growth of wild type Ras-transformed cells. Nck and Grb4 therefore appear to be functionally redundant.

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

R-Ras is regulated by activators and effectors distinct from those that control Ras function.

Like Ras, constitutively activated mutants of the Ras-related protein R-Ras cause tumorigenic transformation of NIH3T3 cells. However, since R-Ras causes a transformed phenotype distinct from that induced by Ras, it is likely that R-Ras controls signaling pathways and cellular processes distinct from those regulated by Ras. To address this possibility, we determined if R-Ras is regulated by activators and effectors distinct from those that regulate Ras function. We observed that Ras guanine nucleotide exchange factors failed to activate R-Ras in vivo, indicating that R-Ras is activated by distinct GEFs. Consistent with this, mutants of R-Ras with mutations analogous to the Ras(15A)/(17N) dominant negative proteins did not antagonize Ras GEF function and lacked the growth inhibitory activity seen with these mutant Ras proteins. Thus, R-Ras, but not Ras, is dispensable for the viability of NIH3T3 cells. Finally, whereas constitutively activated Ras can overcome the growth inhibitory action of the Ras(17N) dominant negative protein via Raf-dependent and -independent activities, transforming mutants of R-Ras failed to do so. This inability was consistent with our observation that Ras-, but not R-Ras-transformed, NIH3T3 cells possessed constitutively upregulated Raf kinase activities. Thus, R-Ras and Ras are regulators of distinct signaling pathways and cellular processes.

Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling.

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.

Interaction of the Nck adapter protein with p21-activated kinase (PAK1).

The p21-activated kinases (PAKs) link G protein-coupled receptors and growth factor receptors (S. Dharmawardhane, R. H. Daniels, and G. M. Bokoch, submitted for publication) to activation of MAP kinase cascades and to cytoskeletal reorganization (M. A. Sells, U. G. Knaus, D. Ambrose, S. Bagrodia, G. M. Bokoch, and J. Chernoff, submitted for publication). The proteins that interact with PAK to mediate its cellular effects and to couple it to upstream receptors are unknown. We describe here a specific interaction of the Nck adapter molecule with PAK1 both in vitro and in vivo. PAK1 and Nck associate in COS-7 and Swiss 3T3 cells constitutively, but this interaction is strengthened upon platelet-derived growth factor receptor stimulation. We show that Nck binds to PAK1 through its second Src homology 3 (SH3) domain, while PAK1 interacts with Nck via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active PAK1 with Nck leads to the phosphorylation of Nck at multiple sites. Association of Nck with PAK1 may serve to link this important regulatory kinase to cell activation by growth factor receptors.

Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors.

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains.

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Guanine nucleotide exchange factors: activators of Ras superfamily proteins.

Members of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP-complexed and inactive GDP-complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP-bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP-bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras-related Rho (Dbl and Vav) proteins. Growth factor-mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10-50 fold and 5-10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras. We have determined that two Ras-related proteins, designated R-Ras and R-Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R-Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R-Ras, transforming activities. Therefore, TC21, but not R-Ras, may be activated by the same extracellular signaling events that activate Ras proteins. Dbl family proteins are believed to function as GEFs and activators of the Ras-related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras-independent activation of MAP kinases and Rho family proteins.